Affiliation: Department of Medicine, University of Chicago, Chicago, Illinois, United States of America.

ABSTRACTTight junctions between intestinal epithelial cells mediate the permeability of the intestinal barrier, and loss of intestinal barrier function mediated by TNF signaling is associated with the inflammatory pathophysiology observed in Crohn's disease and celiac disease. Thus, factors that modulate intestinal epithelial cell response to TNF may be critical for the maintenance of barrier function. TNF alpha-induced protein 3 (TNFAIP3) is a cytosolic protein that acts in a negative feedback loop to regulate cell signaling induced by Toll-like receptor ligands and TNF, suggesting that TNFAIP3 may play a role in regulating the intestinal barrier. To investigate the specific role of TNFAIP3 in intestinal barrier function we assessed barrier permeability in TNFAIP3(-/-) mice and LPS-treated villin-TNFAIP3 transgenic mice. TNFAIP3(-/-) mice had greater intestinal permeability compared to wild-type littermates, while villin-TNFAIP3 transgenic mice were protected from increases in permeability seen within LPS-treated wild-type littermates, indicating that barrier permeability is controlled by TNFAIP3. In cultured human intestinal epithelial cell lines, TNFAIP3 expression regulated both TNF-induced and myosin light chain kinase-regulated tight junction dynamics but did not affect myosin light chain kinase activity. Immunohistochemistry of mouse intestine revealed that TNFAIP3 expression inhibits LPS-induced loss of the tight junction protein occludin from the apical border of the intestinal epithelium. We also found that TNFAIP3 deubiquitinates polyubiquitinated occludin. These in vivo and in vitro studies support the role of TNFAIP3 in promoting intestinal epithelial barrier integrity and demonstrate its novel ability to maintain intestinal homeostasis through tight junction protein regulation.

Mentions:
To assess the direct role of IEC expression of TNFAIP3 in barrier function we generated villin-TNFAIP3 mice that constitutively overexpress a TNFAIP3 transgene in the IECs of the small intestine, cecum and colon (Figures S1, S2). Villin-TNFAIP3 mice are healthy and display normal growth and reproduction up to 6 months of age. We stimulated inflammation in WT and villin-TNFAIP3 mice by injecting LPS into the peritoneal cavity 45 minutes prior to euthanasia. LPS induces a systemic inflammatory response affecting a variety of cell types including IECs, and its short-term effects are mediated largely by the induction of TNF release from innate immune cells, as well as enterocytes in vitro [40]–[41]. LPS induces endogenous TNF release which, when overproduced by intestinal epithelial cells, causes an IBD pathology [42]. The LPS challenge induced greater transepithelial flux of FITC-dextran across the ileum in explanted loops from WT mice compared to villin-TNFAIP3 mice, indicating that epithelial-specific overexpression of TNFAIP3 promotes resistance to inflammatory disruption of the intestinal barrier (Figure 3). Occludin is removed from apical sites of the epithelium and internalized in LPS-treated mice, and recent studies implicate occludin relocation by endocytosis in TNF-induced barrier permeability [14], [43]. Similar to these previously published reports, we observed occludin loss from the apical surface and an overall diminution of occludin intensity in IEC following administration of LPS to WT mice; however, occludin loss was markedly reduced in the intestine of LPS-treated villin-TNFAIP3 mice (Figure 4). This prevention of occludin redistribution suggests a mechanism by which TNFAIP3 affects the structure or integrity of tight junctions in IECs. Together these results demonstrate that TNFAIP3 is required for the maintenance of intestinal barrier function and that TNFAIP3 plays an important role in modulating epithelial permeability, in part by controlling occludin localization or stability in IEC.

Mentions:
To assess the direct role of IEC expression of TNFAIP3 in barrier function we generated villin-TNFAIP3 mice that constitutively overexpress a TNFAIP3 transgene in the IECs of the small intestine, cecum and colon (Figures S1, S2). Villin-TNFAIP3 mice are healthy and display normal growth and reproduction up to 6 months of age. We stimulated inflammation in WT and villin-TNFAIP3 mice by injecting LPS into the peritoneal cavity 45 minutes prior to euthanasia. LPS induces a systemic inflammatory response affecting a variety of cell types including IECs, and its short-term effects are mediated largely by the induction of TNF release from innate immune cells, as well as enterocytes in vitro [40]–[41]. LPS induces endogenous TNF release which, when overproduced by intestinal epithelial cells, causes an IBD pathology [42]. The LPS challenge induced greater transepithelial flux of FITC-dextran across the ileum in explanted loops from WT mice compared to villin-TNFAIP3 mice, indicating that epithelial-specific overexpression of TNFAIP3 promotes resistance to inflammatory disruption of the intestinal barrier (Figure 3). Occludin is removed from apical sites of the epithelium and internalized in LPS-treated mice, and recent studies implicate occludin relocation by endocytosis in TNF-induced barrier permeability [14], [43]. Similar to these previously published reports, we observed occludin loss from the apical surface and an overall diminution of occludin intensity in IEC following administration of LPS to WT mice; however, occludin loss was markedly reduced in the intestine of LPS-treated villin-TNFAIP3 mice (Figure 4). This prevention of occludin redistribution suggests a mechanism by which TNFAIP3 affects the structure or integrity of tight junctions in IECs. Together these results demonstrate that TNFAIP3 is required for the maintenance of intestinal barrier function and that TNFAIP3 plays an important role in modulating epithelial permeability, in part by controlling occludin localization or stability in IEC.

Bottom Line:
In cultured human intestinal epithelial cell lines, TNFAIP3 expression regulated both TNF-induced and myosin light chain kinase-regulated tight junction dynamics but did not affect myosin light chain kinase activity.We also found that TNFAIP3 deubiquitinates polyubiquitinated occludin.These in vivo and in vitro studies support the role of TNFAIP3 in promoting intestinal epithelial barrier integrity and demonstrate its novel ability to maintain intestinal homeostasis through tight junction protein regulation.

Affiliation:
Department of Medicine, University of Chicago, Chicago, Illinois, United States of America.

ABSTRACTTight junctions between intestinal epithelial cells mediate the permeability of the intestinal barrier, and loss of intestinal barrier function mediated by TNF signaling is associated with the inflammatory pathophysiology observed in Crohn's disease and celiac disease. Thus, factors that modulate intestinal epithelial cell response to TNF may be critical for the maintenance of barrier function. TNF alpha-induced protein 3 (TNFAIP3) is a cytosolic protein that acts in a negative feedback loop to regulate cell signaling induced by Toll-like receptor ligands and TNF, suggesting that TNFAIP3 may play a role in regulating the intestinal barrier. To investigate the specific role of TNFAIP3 in intestinal barrier function we assessed barrier permeability in TNFAIP3(-/-) mice and LPS-treated villin-TNFAIP3 transgenic mice. TNFAIP3(-/-) mice had greater intestinal permeability compared to wild-type littermates, while villin-TNFAIP3 transgenic mice were protected from increases in permeability seen within LPS-treated wild-type littermates, indicating that barrier permeability is controlled by TNFAIP3. In cultured human intestinal epithelial cell lines, TNFAIP3 expression regulated both TNF-induced and myosin light chain kinase-regulated tight junction dynamics but did not affect myosin light chain kinase activity. Immunohistochemistry of mouse intestine revealed that TNFAIP3 expression inhibits LPS-induced loss of the tight junction protein occludin from the apical border of the intestinal epithelium. We also found that TNFAIP3 deubiquitinates polyubiquitinated occludin. These in vivo and in vitro studies support the role of TNFAIP3 in promoting intestinal epithelial barrier integrity and demonstrate its novel ability to maintain intestinal homeostasis through tight junction protein regulation.